3T3-L1 Fibroblasts Research Articles

Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.

Chitosan Nanoparticles Containing Lipoic Acid with Antioxidant Properties as a Potential Nutritional Supplement.

Simple SummaryAlfa-lipoic acid (ALA) is an important antioxidant that could be added to animal feed as a nutritional supplement. To improve its stability in the digestive system, ALA was encapsulated in chitosan nanoparticles. The nanoparticles containing ALA were stable in stomach-like conditions and were able to cross the intestinal barrier. Chitosan-based nanoparticles seem to be an attractive administration method for antioxidants, or other sensible additives, in food.The addition of the antioxidant α-lipoic acid (ALA) to a balanced diet might be crucial for the prevention of comorbidities such as cardiovascular diseases, diabetes, and obesity. Due to its low half-life and instability under stomach-like conditions, α-lipoic acid was encapsulated into chitosan nanoparticles (Ch-NPs). The resulting chitosan nanoparticles containing 20% w/w ALA (Ch-ALA-NPs) with an average diameter of 44 nm demonstrated antioxidant activity and stability under stomach-like conditions for up to 3 h. Furthermore, fluorescent Ch-ALA-NPs were effectively internalized into 3T3-L1 fibroblasts and were able to cross the intestinal barrier, as evidenced by everted intestine in vitro experiments. Thus, chitosan-based nanoparticles seem to be an attractive administration method for antioxidants, or other sensible additives, in food.

Mintaimycins, a Group of Novel Peptide Metabolites from Micromonospora sp. C-3509.

A group of peptide metabolites (1–4), designated as mintaimycins, were isolated from Micromonospora sp. C-3509. The planar structures of mintaimycins were determined by combination of mass spectrometry, 1D and 2D NMR spectroscopy, and the stereochemistry of mintaimycins were partially resolved by Marfey’s or Mosher’s method. Mintaimycins featured a central β-methylphenylalanine or phenylalanine linked at its amino group with 5-methyl-2-hexenoic acid, and at its carboxyl group with 5-hydroxy-norleucine or leucine that combined a derivative of hexanoic acid or 4-methylpentanoic acid. Mintaimycin A1 (1), the principal component, was found to exhibit the biological activity of inducing pre-adipocyte differentiation of 3T3-L1 fibroblast cells at 10.0 μmol/L.

The Preliminary Evaluation of Epigenetic Modifications Regulating the Expression of IL10 in Insulin-Resistant Adipocytes.

A higher level of IL10 expression in obesity and insulin resistance was observed in both human and mouse WAT. In our research, we analyzed the influence of insulin resistance on epigenetic modification within the promoter region IL10 gene and the potential influence of these modifications on its expression. Studies were performed using two cell models for the analysis: human, preadipocytes derived from adipose (visceral and subcutaneous) tissues and murine 3T3-L1 fibroblasts. We demonstrated a significant increase in the IL10 expression level, IL10 promoter region methylation, and histone 3 epigenetic modifications: H3K4me and H3K9/14ac, in insulin resistance cells (IR) from SAT cell culture. In IR cells from VAT cell culture, we observed decreased IL10 expression with a simultaneous increase of IL10 promoter region methylation. In IR cells from 3T3L1 cell culture, we observed the increased expression of IL10 as well as the decreased levels of methylation in the IL10 promoter region and histone methylation (H3K4me) and acetylation (H3K9/14ac). The presented analyses suggest a potential impact of epigenetic modifications on gene expression and a potential mutual influence of epigenetic modifications on each other or the activation of specific epigenetic regulation at a different stage of the development of insulin resistance in cells.

BAX But Not BCL2 Is Necessary for Apoptosis in Neuroblastoma Cells Treated With Casiopeína® IIIia.

The emerging antineoplastics Casiopeínas® induce uncoupling of the respiratory chain, production of reactive oxygen species (ROS), entry of Bax into mitochondria, and exit of Ca2+ and Bcl-2 from them, leading to apoptosis. This study aimed to elucidate whether BAX and BCL2 are necessary for apoptosis. We silenced BAX and BCL2 by CRISPR-Cas9, assessed ROS and calcium retention capacity (CRC) by spectrofluorometry, and caspase-3 with inmunoblotting in neuroblastoma (NB) cells and 3T3-L1 fibroblasts treated with cisplatin and Casiopeína IIIia (CasIIIia). We observed an increase in O2•- production only in BCL2KO NB cells treated with cisplatin (three-fold) and CasIIIia (five-fold), whereas the production of H2O2 in BCL2KO NB cells treated with cisplatin and CasIIIia increased five-fold and three-fold, respectively. The baseline calcium-retention capacity (CRC) was 1.7 relative fluorescence units (RFU) in both cell types. In BAXKO, cisplatin and CasIIIia increased CRC to ~2.3 RFU, and in BCL2KO, they decreased CRC to ~1.1 RFU. We did not detect caspase-3 in BAXKO NB cells. Only BAX is essential for CasIIIia-induced apoptosis.

Adipocyte GPX4 protects against inflammation, hepatic insulin resistance and metabolic dysregulation.

Metabolic inflammation is a hallmark of obesity and related disorders, afflicting substantial morbidity and mortality to individuals worldwide. White visceral and subcutaneous adipose tissue not only serves as energy storage but also controls metabolism. Adipose tissue inflammation, commonly observed in human obesity, is considered a critical driver of metabolic perturbation while molecular hubs are poorly explored. Metabolic stress evoked by e.g. long-chain fatty acids leads to oxidative perturbation of adipocytes and production of inflammatory cytokines, fuelling macrophage infiltration and systemic low-grade inflammation. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation, accumulation of oxygen-specific epitopes and cell death, collectively referred to as ferroptosis. Here, we explore the function of adipocyte GPX4 in mammalian metabolism. We studied the regulation and function of GPX4 in differentiated mouse adipocytes derived from 3T3-L1 fibroblasts. We generated two conditional adipocyte-specific Gpx4 knockout mice by crossing Gpx4fl/fl mice with Adipoq-Cre+ (Gpx4-/-AT) or Fabp4-Cre+ (Gpx4+/-Fabp4) mice. Both models were metabolically characterized by a glucose tolerance test and insulin resistance test, and adipose tissue lipid peroxidation, inflammation and cell death were assessed by quantifying oxygen-specific epitopes, transcriptional analysis of chemokines, quantification of F4/80+ macrophages and TUNEL labelling. GPX4 expression was induced during and required for adipocyte differentiation. In mature adipocytes, impaired GPX4 activity spontaneously evoked lipid peroxidation and expression of inflammatory cytokines such as TNF-α, interleukin 1β (IL-1β), IL-6 and the IL-8 homologue CXCL1. Gpx4-/-AT mice spontaneously displayed adipocyte hypertrophy on a chow diet, which was paralleled by the accumulation of oxygen-specific epitopes and macrophage infiltration in adipose tissue. Furthermore, Gpx4-/-AT mice spontaneously developed glucose intolerance, hepatic insulin resistance and systemic low-grade inflammation, when compared to wildtype littermates, which was similarly recapitulated in Gpx4+/-Fabp4 mice. Gpx4-/-AT mice exhibited no signs of adipocyte death. Adipocyte GPX4 protects against spontaneous metabolic dysregulation and systemic low-grade inflammation independent from ferroptosis, which could be therapeutically exploited in the future.

Trinuclear silver(I) complex of non-steroidal anti-inflammatory drug naproxen: Synthesis, characterization, and in vitro cytotoxicity

Herein, a new silver(I) complex with the non-steroidal anti-inflammatory drug naproxen and nitrogen donor 3-picoline ligands was synthesized, characterized, and subsequently tested for its cytotoxicity against different types of cancer cell lines. Elemental analysis, Fourier transform infrared spectroscopy, thermal, and proton nuclear magnetic resonance techniques showed that the molecular formula of the prepared complex is bis(3-picoline)tris(m-naproxenato)trisilver(I) and naproxen ligands bind to silver ions in a bridging bidentate mode. 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) results revealed that silver salts and naproxen alone showed quite weak cytotoxic activity against human breast adenocarcinoma (MDA-MB-453), lung adenocarcinoma (A-549), and colorectal adenocarcinoma (HT-29) cell lines (IC50 > 250 µM), whereas the complex displayed dose dependent cytotoxicity against the aforementioned cell lines. The highest cytotoxicity was observed on MDA-MB-453 cells with an IC50 value of 11.73 µM. Moreover, the complex showed higher selectivity against the cancer cell lines compared to fibroblast 3T3-L1 cells. This study provides preliminary scientific data on the complex for further elucidation of its anticancer mechanism of action.

Antioxidant Activity of Silver-containing Bionanocompositions Based on Humic Substances in Cell Culture

Introduction. Silver nanoparticles are promising agents for suppressing resistant strains of microorganisms and accelerating the purulent wounds healing. Oxidative stress disrupts normal wound healing processes, which leads to the formation of chronic non-healing wounds. Therefore, the determination of the ability of new wound healing agents to decrease the production of reactive oxygen species is a relevant task.Aim. The aim of the current study was to investigate the effect of silver-containing bionanocompositions based on humic substances on the basal and tert-butyl hydroperoxide-stimulated production of reactive oxygen species at the normal fibroblasts 3T3-L1 cell culture in vitro.Materials and methods. The study was carried out on 7 samples of initial humic substances and biomaterials with silver nanoparticles synthesized in the Laboratory of Natural Humic Systems, Faculty of Chemistry, Moscow State University named after M. V. Lomonosov. The intracellular production of reactive oxygen species was assessed using a 2,7-dichlorodihydrofluorescein diacetate fluorescent probe. Cells were cultured with samples for 24 h; tret-butyl hydroperoxide was used to stimulate the production of reactive oxygen species. Detection was performed fluorometrically using a microplate reader.Results and discussion. The most pronounced antioxidant activity was demonstrated by three samples of biomaterials with silver nanoparticles ultradispersed in humic substances matrices (CHS-AgNPs, CHP-AgNPs and CHE-AgNPs), which allows us to consider them as the most promising pharmaceutical agents for the treatment of purulent-inflammatory processes. The most probable mechanism of the high antioxidant activity of the studied biomaterials in relation to intracellular reactive oxygen species is the intrinsic activity of humic substances to bind reactive oxygen species, while silver nanoparticles in biomaterials catalyze the reduction processes of their interaction with reactive oxygen species.Conclusion. For the studied samples of biomaterials with silver nanoparticles ultradispersed in matrices of humic substances pronounced antioxidant activity was shown. Together with antibacterial properties, it makes it possible to consider them as potential agents for purulent wounds healing accelerating.

Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells.

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

Anti-lipid droplets accumulation effect of Annona montana (mountain soursop) leaves extract on differentiation of preadipocytes.

The Annona genus is a member of Annonaceae, one of the largest families of plants across tropical and subtropical regions. This family has been used in several ethnomedicinal practices to treat a multitude of human diseases. However, the molecular mechanism underlying its effect on the lipid droplet formation and on the expression of adipogenic markers of this plant remain to be investigated. In this study, we examined whether the extracts from the aerial part of Annona montana affect in vitro differentiation of preadipocytes. For our investigations, both mouse embryo fibroblast 3T3-L1 and normal human primary subcutaneous preadipocytes were incubated with Annona montana extracts (-and its subfractions-) and then analyzed on preadipocyte differentiation, lipid content, lipid droplet size and number, the expression of adipogenic-specific transcriptional factors, as well as cell survival. From our examinations, we found the Annona montana ethyl acetate extract to exhibit a potent inhibitory effect on adipogenesis, without affecting cell survival, in a dose-dependent manner. Such inhibitory effects included a significant decrease in the accumulation of lipid content by both a dramatic reduction of size and number of lipid droplets. This extract strongly attenuated the expression of PPARγ and HMGB2. It also inhibited the expression of CEBPα, FAS, and Akt without influencing Erk1/2 activities. Our findings suggest that specifically, the Annona montana ethyl acetate extract has a prominent inhibitory effect in cellular pathways of adipocyte differentiation by modulating specific gene expression, which is known to perform a pivotal role during adipogenesis.

A bis indole analog molecule fluorescent probe for living cell staining

In this study, the fluorescent probe property of a bis indole compound (5,14-dimethyl-5,6,7,8,9,14-hexahydroindolo [2 ', 3′: 3,4] cycloocta [1,2-b] indole), a Caulerpin derivative, which is a member of the indole family, was investigated. For this purpose, firstly, the fluorescence character of the molecule was investigated by absorption and fluorescence spectroscopy techniques in both solvent and surfactant environments. Afterwards, the cytotoxicity of the molecule was tested on the human colon cancer HT-29 cells and breast cancer MCF-7 cells as well as mouse fibroblast 3T3-L1 cells. After determining that the molecule has no cytotoxic effect against all the tested cell lines by 100 µM, the cell staining feature of the probe was examined. The obtained data showed us that the molecule, whose confocal microscopy images were taken at 360 nm excitation wavelength, stained the cell cytoplasm. In addition, optical absorption spectra, oscillator strength (f), transition configuration and transition characteristics of the Ars-In in different solvents were investigated at the theoretical level using Time Dependent-Density Functional Theory (TD-DFT). In conclusion, these results confirm that this indole derivative molecule could be evaluated as an effective fluorescence probe for biological systems.

Phospholipid Derivatives of Cinnamic Acid Restore Insulin Sensitivity in Insulin Resistance in 3T3-L1 Adipocytes.

Background: Insulin resistance (IR) is a condition in which the physiological amount of insulin is insufficient to evoke a proper response of the cell, that is, glucose utilization. Metformin is the first choice for therapy, thanks to its glycemic efficacy and general tolerability. In addition, various natural compounds from plant extracts, spices, and essential oils have been shown to provide health benefits regarding insulin sensitivity. In the present study, we analyzed the effect of phospholipid derivatives of selected natural aromatic acids on insulin action and their potential use to overcome insulin resistance. Methods: The 3T3-L1 fibroblasts were differentiated into mature adipocytes; next, insulin resistance was induced by palmitic acid (16:0). Cells were further cultured with phenophospholipids at appropriate concentrations. To assess insulin sensitivity, we measured the insulin-stimulated glucose uptake, using a glucose uptake test. Results: We showed that cinnamic acid (CA) and 3-methoxycinnamic acid (3-OMe-CA) restored the proper insulin response. However, 1,2-dicinnamoyl-sn-glycero-3-phosphocholine (1,2-diCA-PC) and 1-cinnamoyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-CA-2-PA-PC) improved insulin sensitivity in insulin-resistant adipocytes even stronger, exhibiting more beneficial effects. Conclusions: The binding of aromatic acids to phosphatidylcholine increases their beneficial effect on insulin sensitivity in adipocytes and expands their potential practical application as nutraceutical health-promoting agents.

Chemical composition and biological activity of Peucedanum dhana A. Ham essential oil

The essential oil was extracted from Peucedanum dhana A. Ham, which grows in Thailand, using a Clevenger apparatus, resulting in an oil yield of 0.76% w/w. Forty-two compounds were identified using gas chromatography-mass spectrometry. The major compounds were trans-piperitol (51.23%), β-pinene (11.72%), o-cymene (11.12%), γ-terpinene (9.21%), and limonene (4.91%). The antimicrobial activity of the P. dhana essential oil was investigated by measuring the inhibition zone diameter, minimum inhibitory concentration (MIC), and minimum microbicidal concentration (MMC). The inhibition zone diameters of P. dhana essential oil (1000 µg/mL) against tested pathogens ranged from 10.70 to 40.80 mm. Significant antimicrobial activity against tested pathogens was obtained, with MIC and MMC values of 62.50–250 µg/mL and 250–1000 µg/mL, respectively. Escherichia coli, Pseudomonas aeruginosa, and Enterobacter aerogenes exposed to P. dhana essential oil at the MIC were analysed by flow cytometry using propidium iodide (PI) and SYTO9 to assess membrane integrity compared to trans-piperitol and β-pinene. After 24 h, treatments with trans-piperitol resulted in the most significant cell membrane alteration and depolarization followed by P. dhana essential oil and β-pinene, respectively. It was demonstrated that the P. dhana essential oil presented antibacterial action against E. coli, P. aeruginosa, and E. aerogenes. The antioxidant activity of P. dhana essential oil was measured using 2,2-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) scavenging activity assays. The IC50 values obtained from the DPPH and ABTS methods were 9.13 and 9.36 mg/mL, respectively. The cytotoxic effect of P. dhana oil was tested against human colonic adenocarcinoma (SW480), human lung adenocarcinoma (A549), cervical cancer (Hela), and murine fibroblast (3T3L1) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The essential oil had cytotoxicity against all cancer cells, with significant cytotoxicity towards SW480 cells. As a control experiment, two pure compounds—trans-piperitol and β-pinene, were also tested for their antimicrobial, antioxidant, and cytotoxic activity. Both compounds showed varied activity in all assays. The results indicate that P. dhana essential oil could be used as a source of functional ingredients in food and pharmaceutical applications.

ZnO nanoparticles stimulate oxidative stress to induce apoptosis of B16F10 melanoma cells: In vitro and in vivo studies

Melanoma is one of the most aggressive skin cancers. However, there remain many limitations in the current clinical treatments of it. Zinc oxide nanoparticles (ZnO NPs) have been considered to be a promising antitumor drug due to their excellent biocompatibility, biodegradability and biofunctionality. In this study, we prepared spherical ZnO NPs with an average diameter of less than 10 nm by a simple chemical method. According to the in vitro cytotoxicity assay, ZnO NPs in a certain concentration range (20–35 μg ml−1) showed significant cytotoxicity to B16F10 melanoma cells, while having little effect on the viability of 3T3L1 fibroblasts. When cultured with B16F10 melanoma cells, ZnO NPs induced the generation of reactive oxygen and mitochondrial superoxide through the release of Zn2+, leading to oxidative stress in the cells, further reducing the mitochondrial membrane potential and decreasing the number of mitochondrial cristae. Furthermore, damaged mitochondria induced the release of apoptosis factors to promote cell apoptosis. FITC-Annexin V/propidium iodide double staining assay was used to analyze different apoptosis stages of B16F10 cells induced by ZnO NPs. A polymer hydrogel (Gel-F127-ZnO NPs) with Pluronic F127 as the carrier of ZnO NPs was fabricated for evaluating the antitumor effect of ZnO NPs in vivo. The in vivo experiment indicated that the tumor recurrence was significantly inhibited in tumor-bearing mice after treated with Gel-F127-ZnO NPs. Conclusively, ZnO NPs showed a strong antitumor effect both in vitro and in vivo.

DLC-Coated Ferroelectric Membranes as Vascular Patches: Physico-Chemical Properties and Biocompatibility

In this paper, the results on the fabrication of ferroelectric membranes as vascular patches with modified surfaces are presented. For the modification of a membrane surface contacting blood, DLC coating was deposited using the pulsed vacuum arc deposition technique. The physico-chemical properties and cytotoxicity of the membranes modified under various conditions were studied. It was found that DLC coatings do not affect membrane microstructure, preserving its crystal structure as well as its high strength and elongation. It was revealed that an increase in the capacitor storage voltage results in the rise in sp2- and sp-hybridized carbon concentration, which makes it possible to control the chemical structure and surface energy of the modified surface. The experiments with 3T3L1 fibroblasts showed no toxic effects of the materials extracts.

Abstract MP32: Diaphanous 1 Regulates Actin Polymerization In Adipocytes: Potential Mechanism Regulating Obesity In Mice

The underlying pathophysiological mechanisms of obesity, a hallmark of metabolic and cardiovascular diseases, are not fully elucidated. Adipocyte hypertrophy contributes to insulin resistance, and is coupled to meaningful alterations in actin dynamics that favor actin polymerization. Diaphanous 1 (Diaph1) is a formin protein that associates with the fast-growing/barded end of actin filaments to regulate actin dynamics. We hypothesized that deletion of Diaph1 or pharmacological regulation of actin polymerization in the context of lipid surplus would prevent obesity, decrease adipocyte lipid load and protect from obesity-associated metabolic disease. We modeled obesity by feeding wild type (Wt) mice and mice globally or with adipocyte-specific deletion of Diaph1 a high fat diet (60%kcal from fat). Body composition was assessed by Dual-energy X-ray absorptiometry (DEXA) and dynamic glucose and insulin tolerance testing was performed to assess metabolic state. In-vitro studies were carried out in adipocytes differentiated from primary pre-adipocytes or 3T3-L1 fibroblasts. Lipid overload was modeled using a mixture of palmitate/oleate (P/O). Actin manipulations were achieved by pharmacologically preventing (Latrunculin B [LatB]) or promoting (Jasplakinolide [Jasp]) F-actin polymerization. Mice globally devoid of, or with adipocyte specific Diaph1 deletion were protected from obesity, glucose intolerance and insulin resistance. Wt adipocytes treated with P/O exhibited pronounced accumulation of triglycerides (Tg) which was absent in adipocytes differentiated from Diaph1 knock out mice. Pharmacological studies in 3T3-L1-derived adipocytes confirmed the P/O-induced Tg accumulation which was prevented by LatB and exacerbated by Jasp. We present the novel finding that adipocyte DIAPH1 is a major player in the pathophysiology of obesity and associated metabolic sequela which results, at least in part, from regulation of adipocyte hypertrophy by preventing actin polymerization. Thus, we propose that actin remodeling is an underlying mechanism for adipocyte hypertrophy in obesity and inhibition of DIAPH1 may represent a potential therapeutic target for the treatment of obesity and related metabolic and cardiovascular disorders.

Synthesis and Study of Antifungal Properties of New Cationic Beta-Glucan Derivatives.

The interaction of positively charged polymers (polycations) with a biological membrane is considered to be the cause of the frequently observed toxicity of these macromolecules. If it is possible to obtain polymers with a predominantly negative effect on bacterial and fungal cells, such systems would have great potential in the treatment of infectious diseases, especially now when reports indicate the growing risk of fungal co-infections in COVID-19 patients. We describe in this article cationic derivatives of natural beta-glucan polymers obtained by reacting the polysaccharide isolated from Saccharomyces boulardii (SB) and Cetraria islandica (CI) with glycidyl trimethyl ammonium chloride (GTMAC). Two synthesis strategies were applied to optimize the product yield. Fungal diseases particularly affect low-income countries, hence the emphasis on the simplicity of the synthesis of such drugs so they can be produced without outside help. The three structures obtained showed selective anti-mycotic properties (against, i.e., Scopulariopsis brevicaulis, Aspergillus brasiliensis, and Fusarium solani), and their toxicity established using fibroblast 3T3-L1 cell line was negligible in a wide range of concentrations. For one of the polymers (SB derivative), using in vivo model of Aspergillus brasiliensis infection in Galleria mellonella insect model, we confirmed the promising results obtained in the preliminary study.

Nanofibrous scaffolds of carboxymethyl guargum potentiated with reduced graphene oxide for in vitro and in vivo wound healing applications

Nanofiber scaffolds mimic the extracellular matrix (ECM) and help in fibroblasts proliferation which is the main constituent for wound healing. This study aims to evaluate the wound healing potential of electrospun nanofibers fabricated by carboxymethyl guargum (CMGG), reduced graphene oxide (rGO) and polyvinyl alcohol. The nanofibers have shown desired properties like excellent porosity and good water holding capacities. The porosity of nanofibers helps in the movement of oxygen to cells and the removal of waste materials and the swelling capacity helps to maintain the moisture content at the wound site. In addition, the in vitro hemocompatibility and wound healing assay have shown excellent results rendering the nanofibers biocompatible. The in vitro fibroblasts (3T3-L1) proliferation was significantly more in rGO/CMGG/PVA nanofibers than CMGG/PVA and cell control. Further, the in vivo wound healing evaluation of these nanofiber dressings in rabbits has shown significant wound closure compared to control and standard. Histology studies revealed the fast collagen formation and re-epithelialization necessary for wound healing among rGO/CMGG/PVA treated rabbits. Therefore, the rGO/CMGG/PVA nanofiber scaffolds can be potential wound dressing candidates and be further evaluated for clinical use.

Alendronate inhibits triglyceride accumulation and oxidative stress in adipocytes and the inflammatory response of macrophages which are associated with adipose tissue dysfunction.

Alendronate, a bisphosphonate used to prevent osteoporosis, stimulates osteogenesis but impairs adipogenesis. Different clinical trials suggest that the incidence of diabetes may be lower in patients treated with alendronate. Taking into account the importance of adipocytes and macrophages of adipose tissue in insulin resistance and type 2 diabetes, it is necessary to evaluate the effect of alendronate in both cell types. In this paper, we investigated the effect of alendronate on the differentiation to adipocytes of 3T3-L1 fibroblasts, the cell line most used to study adipogenesis, and also its effect on lipid content and oxidative stress in mature adipocytes as well as on the inflammatory response of macrophages. We found that alendronate inhibits differentiation of 3T3-L1 fibroblasts to adipocytes in keeping with reports in other cell lines. On the other hand, treatment of 3T3-L1 adipocytes with alendronate was able to decrease triglyceride content and to prevent H2O2-induced lipid peroxidation which was evaluated as an indicator of oxidative stress. In addition, it was found that activation of RAW 264.7 macrophages to a pro-inflammatory M1 type is inhibited by this bisphosphonate. These results suggest that alendronate may contribute to prevent adipocyte excessive enlargement and the induction of oxidative stress in 3T3-L1 adipocytes as well as the activation of macrophages to a pro-inflammatory M1 type, which are events associated with adipose tissue dysfunction and insulin resistance. In this study, we unraveled the underlying mechanisms of events that were previously observed in clinical trials.

Bioengineering for curcumin loaded carboxymethyl guargum/reduced graphene oxide nanocomposites for chronic wound healing applications

Biomimetic scaffolds engineering for improved collagen, epithelial cutaneous and fibrous tissue regeneration remains challenging for wound healing. To address these issues, this study aimed to report on the fabrication and characterization of electrospun of carboxymethyl guargum (CMGG), reduced graphene oxide (rGO) nanocomposite dressings loaded with curcumin for chronic wound healing applications. SEM and XRD examined the morphology of nanofibers and resulted in excellent porosity. TGA and FT-IR were done, which revealed the nanofibers' thermal and chemical interactions. CMGG, rGO nanocomposite with curcumin was investigated for in-vitro wound healing assay by scratch wound healing model using 3T3 L1 fibroblast cell lines and conducted in vitro drug-releasing studies. These nanocomposites showed 100% wound closure by the proliferation of fibroblast cell lines 3T3-L1 within 48 h and showed controlled drug release. Further, in vivo results also showed that the CMGG, rGO nanocomposite with curcumin has the potential wound healing effects. Histological studies showed that the CMGG, rGO nanocomposite with curcumin has the potential for wound healing, which indicates that the biomimetic CMGG nanofibers have an excellent healing effect on chronic wounds.